Intravenous administration of anti-α4β7 monoclonal antibody in macaques decreases simian immunodeficiency virus (SIV) vaginal infection and reduces gut SIV loads. Because of potential side effects of systemic administration, a prophylactic strategy based on mucosal administration of anti-α4β7 antibody may be safer and more effective. With this in mind, we developed a novel intravaginal formulation consisting of anti-α4β7 monoclonal antibody–conjugated nanoparticles (NPs) loaded in a 1% hydroxyethylcellulose (HEC) gel (NP-α4β7 gel). When intravaginally administered as a single dose in a rhesus macaque model, the formulation preferentially bound to CD4+ or CD3+ T cells expressing high levels of α4β7, and occupied ~40% of α4β7 expressed by these subsets and ~25% of all cells expressing α4β7. Blocking of the α4β7 was restricted to the vaginal tract without any changes detected systemically.